Numerous biological substances are quantitatively or semiquantitatively determined by immunological methods. Radioimmunoassay (RIA) opened a new generation of trace determination techniques and permitted a degree of sensitivity into the molecular range, not hitherto attainable. RIA techniques are presently being displaced quite extensively, however, nonradioactive determining methods are needed which permit an approximately equal degree of sensitivity, but avoid the problems inherent in handling radioactive materials. One such method is the widely used fluoroimmunoassay, which is well known and is extensively described in the published literature. See, for example, Davis et al, MICROBIOLOGY, 2nd edition, Harper & Row, 1973, pp. 397 et seq.; Cooper, THE TOOLS OF BIOCHEMISTRY, Wiley-Interscience, New York, 1977; and Iesen, IMMUNOLOGY, Harper & Row, 1974.
Fluoroimmunoasay techniques using pulsed light, i.e., a strobe light or photoflash type light, referred to as pulsed fluoroimmunoassay (PFIA), have been described in my U.S. Pat. No. 4,133,873 and my German Patent Application corresponding thereto, No. P 25 23 209.1. PFIA displays numerous advantages as compared with other isotopic and nonisotopic immunoassay methods. The literature has also reported that fluorescent molecules, when excited by polarized light, emit luminous energy which, in its polarization value, decisively depends upon the molecular size of the species which fluoresced. The degree of polarization also depends upon other parameters, such as, for example, the number and type of these molecules, the state of the molecules, i.e., whether or not the molecules are bound at one position or are unbonded to one another, etc. See the foregoing references and Parker, C.W., in Weir, HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, third edition, Volume I, pp. 18.1 et seq.
With the formation of the antigen-antibody complex, the molecular size of the species is changed in all immunological reactions. As a result of this change in size in the immunological reaction, a fluorescent tagged immunological substance undergoes a change in its fluorescence polarization characteristics, i.e., a fluorescent tagged antigen fluoresces differently and, in particular, has a different degree of rotation in its unbound condition as compared with its condition when bound to an antibody. Similarly, a fluorescent tagged unbound antibody behaves differently than the same antibody bound to an antigen.